Non-natural ribonuclease conjugates as cytotoxic agents

ABSTRACT

The present invention is directed toward the delivery of a toxic protein to pathogenic cells, particularly cancer cells. In preferred embodiments, the toxic protein is a ribonuclease that has been modified to make it toxic to target cells and that can be conjugated to a target cell-specific delivery vector, such as an antibody, for delivery to pathogenic cells.

The present Application claims priority to U.S. Provisional ApplicationSer. No. 60/561,609 filed Apr. 13, 2004, which is herein incorporated byreference.

FIELD OF THE INVENTION

The present invention is directed toward the delivery of a toxic proteinto pathogenic cells, particularly cancer cells. In preferredembodiments, the toxic protein is a ribonuclease that has been modifiedto make it toxic to target cells and that can be conjugated to a targetcell-specific delivery vector, such as an antibody, for delivery topathogenic cells.

BACKGROUND OF THE INVENTION

The term “chemotherapy” simply means the treatment of disease withchemical substances. The father of chemotherapy, Paul Ehrlich, imaginedthe perfect chemotherapeutic as a “magic bullet;” such a compound wouldkill invading organisms or cells without harming the host. Whilesignificant progress has been made in identifying compounds that kill orinhibit cancer cells and in identifying methods of directing suchcompounds to the intended target cells, the art remains in need ofimproved anti-cancer compounds.

SUMMARY OF THE INVENTION

The present invention relates to the use (e.g., therapeutic use,diagnostic use, research use) of proteins to target cancers and otherdiseases and conditions (e.g., viral or pathogen infections) whereselectively killing pathogenic cells is desired. For example, in someembodiments, the present invention relates to the production anddelivery of a cytotoxic protein to pathogenic cells such as tumor cellsor virus-infected cells. The ribonuclease can also be used to degradepathogenic RNA outside of the cell. In some preferred embodiments, thepresent invention provides the use of ribonuclease proteins (e.g., humanribonuclease proteins) that are altered in their amino acid sequence(i.e., non-natural) to make them cytotoxic. In some embodiments, thesemutated proteins are specifically delivered to pathogenic cells byconjugation to targeting vectors (e.g., human or humanized protein) thatare specific for or at least partially selective for the pathogenictarget cells. Such targeting vectors include, but are not limited to,antibodies, receptors, ligands, peptides, nucleic acids, lipids,polymers, small molecules, and synthetic compounds. In some embodiments,mutant ribonuclease genes are delivered as DNA or RNA via expressionvectors. The ribonuclease genes may be expressed alone, or may beexpressed as chimerical conjugates of the ribonuclease gene with acell-specific targeting moiety.

The present invention also provides methods comprising the delivery ofthe cytotoxic ribonucleases under conditions that minimize or eliminatethe human immune response against the proteins and delivery vectors.This present invention further provides methods for selective inhibitionof cellular growth and/or viral replication in target cells through theaction of the mutated ribonucleases.

Thus, in some embodiments, the present invention provides a novel familyof proteins for treating, characterizing, or understanding disease. Insome embodiments, the compositions of the present invention are usedtherapeutically, alone or in combination with other compounds orinterventions (e.g., to augment existing therapies for treatment ofhuman cancers).

Thus, in some embodiments, the present invention provides a compositioncomprising a non-natural ribonuclease (e.g., human ribonuclease)conjugated to a cell-specific targeting moiety, wherein the ribonucleaseis configured to kill the cell. In some embodiments, the non-naturalhuman ribonuclease comprises a non-natural human ribonuclease one (RNase1). Examples of suitable non-natural human RNase 1 compounds include,but are not limited to, those having a variant sequence compared to anatural ribonuclease one as shown in Table 1. TABLE 1 N88C L86E, N88R,G89D, R91D R4C, L86E, N88R, G89D, R91D, V118C L86E, N88C, R91D R4C,L86E, N88C, R91D, V118C R4C, N88C, V118C K7A, L86E, N88C, R91D K7A,L86E, N88R, G89D, R91D R4C, K7A, L86E, N88C, R91D, V118C R4C, K7A, L86E,N88R, G89D, R91D, V118C

The present invention further provides variants of such sequences.Exemplary variants are provided in Tables 2 and 3, below, as well as inExample 2. Additional variants that have the desired function are alsowithin the scope of the invention. TABLE 2 Human ribonuclease I aminoacid modifications for increased cytotoxicity Amino Acid Amino AcidPosition Identity Amino Acids Substitution 7 Lysine (K) Glycine (G),Alanine (A), Aspartatic acid (D), Glutamatic acid (E), Phenylalanine(F), Tryptophan (W) 85 Arginine (R) Aspartatic acid (D), Glutamatic acid(E), Phenylalanine (F), Tryptophan (W), Glycine (G), Alanine (A) 86Leucine (L) Aspartatic acid (D), Glutamatic acid (E), Phenylalanine (F),Lysine (K), Arginine (R), Tryptophan (W) 87 Threonine (T) Leucine (L),Phenylalanine (F), Tyrosine (Y), Tryptophan (W) 88 Asparagine (N) Lysine(K), Arginine (R), Leucine (L), Aspartic acid (D), Glutamatic acid (E),Phenylalanine (F), Tyrosine (Y), Tryptophan (W) 89 Glycine (G) Lysine(K), Arginine (R), Leucine (L), Aspartic acid (D), Glutamatic acid (E),Phenylalanine (F), Tyrosine (Y), Tryptophan (W) 90 Serine (S)Phenylalanine (F), Tryptophan (W), Aspartatic acid (D), Glutamatic acid(E), Lysine (K), Arginine (R) 91 Arginine (R) Aspartatic acid (D),Glutamatic acid (E), Phenylalanine (F), Tryptophan (W) 92 Tyrosine (Y)Glycine (G), Alanine (A), Lysine (K), Arginine (R), Aspartic acid (D),Glutamatic acid (E) 93 Proline (P) Leucine (L), Phenylalanine (F),Tyrosine (Y), Tryptophan (W), Lysine (K), Arginine (R), Aspartic acid(D), Glutamatic acid (E) 94 Asparagine (N) Lysine (K), Arginine (R),Leucine (L), Aspartic acid (D), Glutamatic acid (E), Phenylalanine (F),Tyrosine (Y), Tryptophan (W)

TABLE 3 Amino Acid Amino Acid Position Identity Amino Acids Substitution1 Lysine (K) Cysteine (C) 2 Glutamic acid (E) Cysteine (C) 3 Serine (S)Cysteine (C) 4 Arginine (R) Cysteine (C) 5 Alanine (A) Cysteine (C) 6Lysine (K) Cysteine (C) 116 Valine (V) Cysteine (C) 117 Proline (P)Cysteine (C) 118 Valine (V) Cysteine (C) 119 Histidine (H) Cysteine (C)120 Phenylalanine (F) Cysteine (C) 121 Aspartate (D) Cysteine (C)

In some embodiments, a plurality of different ribonucleases and/ortargeting moieties are provided in a composition (e.g., a kit, apharmaceutical preparation, etc.)

The present invention is not limited by the nature of or location of thetarget cell. In some embodiments, the cell is a cancer cell, a cell thatexpresses a marker associated with viral infection, a cell that isassociated with an inflammatory response, and a cell is associated withan autoimmune disease (e.g., a cell expressing markers or otherwisecharacterized as aberrantly failing to undergo cell death or presentingautoantigens). In some embodiments, the cell resides in vitro (e.g., inculture). In other embodiments, the cell resides in vivo (e.g., in atissue, as a transplanted cell, in a test animal, in a subject suspectedof or diagnosed as having a disease or condition—e.g., cancer).

In some preferred embodiments, the ribonuclease is conjugated to thecell-specific targeting moiety by a linker. The present invention is notlimited by the nature of the linker. Linkers suitable for use with thepresent invention include, but are not limited to, linkers attached to anon-native cysteine of the ribonuclease, non-cleavable linkers,cleavable linker, and linkers attached within a loop region of theribonuclease corresponding to amino acids 84-95 of bovine ribonucleaseA.

In some embodiments, the ribonuclease is made as a fusion protein with adisease-specific protein, such as an antibody or antibody fragment.Those skilled in the art recognize that the fusion can be created usingcDNA and standard molecular biology techniques.

The present invention is not limited by the nature of the cell-specifictargeting moiety. Targeting moieties include, but are not limited to,immunoglobulins (e.g., antibodies, humanized or partially humanizedantibodies, antibody fragments, etc.), proteins, peptides, receptorligands, aptamers, small molecules, nucleic acid molecules, lipids, etc.

In some embodiments, the components of the composition (e.g., theribonuclease, the cell specific-targeting moieity) are provided to acell, alone or together via an expression vector, such that thecomponents are produce within a cell of a subject or produced within acell provided to the subject (e.g., through ex vivo transfectionfollowed by transplantation).

The present invention further provides methods of killing cell using anyof the compositions discussed herein.

DESCRIPTION OF FIGURES

FIG. 1A shows a graph demonstrating the growth inhibiting effect ofQBI-119 on tumor volume (A549 cells) over a number of days, and

FIG. 1B shows a graph depicting the lack of toxicity of QBI-119.

FIG. 2A shows a graph demonstrating the growth inhibiting effect ofQBI-119 on tumor volume (Bx-PC-3 cells) over a number of days, and FIG.2B shows a graph depicting the lack of toxicity of QBI-119.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “immunoglobulin” or “antibody” refer toproteins that bind a specific antigen. Immunoglobulins include, but arenot limited to, polyclonal, monoclonal, chimeric, and humanizedantibodies, Fab fragments, F(ab′)₂ fragments, and includesimmunoglobulins of the following classes: IgG, IgA, IgM, IgD, IbE, andsecreted immunoglobulins (slg). Immunoglobulins generally comprise twoidentical heavy chains and two light chains. However, the terms“antibody” and “immunoglobulin” also encompass single chain antibodiesand two chain antibodies.

As used herein, the term “antigen binding protein” refers to proteinsthat bind to a specific antigen. “Antigen binding proteins” include, butare not limited to, immunoglobulins, including polyclonal, monoclonal,chimeric, and humanized antibodies; Fab fragments, F(ab′)₂ fragments,and Fab expression libraries; and single chain antibodies.

The term “epitope” as used herein refers to that portion of an antigenthat makes contact with a particular immunoglobulin.

When a protein or fragment of a protein is used to immunize a hostanimal, numerous regions of the protein may induce the production ofantibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as “antigenic determinants”. An antigenic determinantmay compete with the intact antigen (i.e., the “immunogen” used toelicit the immune response) for binding to an antibody.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

As used herein, the terms “non-specific binding” and “backgroundbinding” when used in reference to the interaction of an antibody and aprotein or peptide refer to an interaction that is not dependent on thepresence of a particular structure (i.e., the antibody is binding toproteins in general rather that a particular structure such as anepitope).

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the term “subject suspected of having cancer” refers toa subject that presents one or more symptoms indicative of a cancer(e.g., a noticeable lump or mass) or is being screened for a cancer(e.g., during a routine physical). A subject suspected of having cancermay also have one or more risk factors. A subject suspected of havingcancer has generally not been tested for cancer. However, a “subjectsuspected of having cancer” encompasses an individual who has received apreliminary diagnosis (e.g., a CT scan showing a mass or increased PSAlevel) but for whom a confirmatory test (e.g., biopsy and/or histology)has not been done or for whom the stage of cancer is not known. The termfurther includes people who once had cancer (e.g., an individual inremission). A “subject suspected of having cancer” is sometimesdiagnosed with cancer and is sometimes found to not have cancer.

As used herein, the term “subject diagnosed with a cancer” refers to asubject who has been tested and found to have cancerous cells. Thecancer may be diagnosed using any suitable method, including but notlimited to, biopsy, x-ray, blood test, and the diagnostic methods of thepresent invention. A “preliminary diagnosis” is one based only on visual(e.g., CT scan or the presence of a lump) and antigen tests (e.g., PSA).

As used herein, the term “subject at risk for cancer” refers to asubject with one or more risk factors for developing a specific cancer.Risk factors include, but are not limited to, gender, age, geneticpredisposition, environmental exposure, and previous incidents ofcancer, preexisting non-cancer diseases, and lifestyle.

As used herein, the term “non-human animals” refers to all non-humananimals including, but are not limited to, vertebrates such as rodents,non-human primates, ovines, bovines, ruminants, lagomorphs, porcines,caprines, equines, canines, felines, aves, etc.

As used herein, the term “gene transfer system” refers to any means ofdelivering a composition comprising a nucleic acid sequence to a cell ortissue. For example, gene transfer systems include, but are not limitedto, vectors (e.g., retroviral, adenoviral, adeno-associated viral, andother nucleic acid-based delivery systems), microinjection of nakednucleic acid, polymer-based delivery systems (e.g., liposome-based andmetallic particle-based systems), biolistic injection, and the like. Asused herein, the term “viral gene transfer system” refers to genetransfer systems comprising viral elements (e.g., intact viruses,modified viruses and viral components such as nucleic acids or proteins)to facilitate delivery of the sample to a desired cell or tissue. Asused herein, the term “adenovirus gene transfer system” refers to genetransfer systems comprising intact or altered viruses belonging to thefamily Adenoviridae.

“Amino acid sequence” and terms such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

The terms “test compound” and “candidate compound” refer to any chemicalor biological entity, pharmaceutical, drug, and the like that is acandidate for use to treat or prevent a disease, illness, sickness, ordisorder of bodily function (e.g., cancer). Test compounds comprise bothknown and potential therapeutic compounds. A test compound can bedetermined to be therapeutic by screening using the screening methods ofthe present invention.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Environmental samplesinclude environmental material such as surface matter, soil, water andindustrial samples. Such examples are not however to be construed aslimiting the sample types applicable to the present invention.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of a polypeptideor precursor (e.g., ribonucleases or ribonuclease conjugates of thepresent invention). The polypeptide can be encoded by a full lengthcoding sequence or by any portion of the coding sequence so long as thedesired activity or functional properties (e.g., enzymatic activity,etc.) of the full-length or fragment are retained. The term alsoencompasses the coding region of a structural gene and the includingsequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb on either end such that the genecorresponds to the length of the full-length mRNA. The sequences thatare located 5′ of the coding region and which are present on the mRNAare referred to as 5′ untranslated sequences. The sequences that arelocated 3′ or downstream of the coding region and that are present onthe mRNA are referred to as 3′ untranslated sequences. The term “gene”encompasses both cDNA and genomic forms of a gene. A genomic form orclone of a gene contains the coding region interrupted with non-codingsequences termed “introns” or “intervening regions” or “interveningsequences.” Introns are segments of a gene that are transcribed intonuclear RNA (hnRNA); introns may contain regulatory elements such asenhancers. Introns are removed or “spliced out” from the nuclear orprimary transcript; introns therefore are absent in the messenger RNA(niRNA) transcript. The mRNA functions during translation to specify thesequence or order of amino acids in a nascent polypeptide.

The term “wild-type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the terms“modified”, “mutant”, and “variant” refer to a gene or gene product thatdisplays modifications in sequence and or functional properties (i.e.,altered characteristics) when compared to the wild-type gene or geneproduct. It is noted that naturally-occurring mutants can be isolated;these are identified by the fact that they have altered characteristicswhen compared to the wild-type gene or gene product. This is in contrastto synthetic mutants that are changes made in a sequence through human(or machine) intervention.

The term “fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion as compared to thenative protein, but where the remaining amino acid sequence is identicalto the corresponding positions in the amino acid sequence deduced from afull-length cDNA sequence. Fragments typically are at least 4 aminoacids long, preferably at least 20 amino acids long, usually at least 50amino acids long or longer, and span the portion of the polypeptiderequired for intermolecular binding of the compositions with its variousligands and/or substrates.

As used herein, the term “purified” or “to purify” refers to the removalof impurities and contaminants from a sample. For example, antibodiesare purified by removal of non-immunoglobulin proteins; they are alsopurified by the removal of immunoglobulin that does not bind an intendedtarget molecule. The removal of non-immunoglobulin proteins and/or theremoval of immunoglobulins that do not bind an intended target moleculeresults in an increase in the percent of target-reactive immunoglobulinsin the sample. In another example, recombinant polypeptides areexpressed in host cells and the polypeptides are purified by the removalof host cell proteins; the percent of recombinant polypeptides isthereby increased in the sample.

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome-binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the term “host cell” refers to any eukaryotic orprokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in a transgenic animal.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed toward the delivery of a toxic proteinto pathogenic cells, particularly cancer cells. The toxic protein mayalso have benefit when delivered to diseased areas without being toxicto the diseased cells. In preferred embodiments, the toxic protein is aribonuclease that has been modified to make it toxic to target cells andthat can be conjugated to a target cell-specific delivery vector, suchas an antibody, for delivery to pathogenic cells.

Preferred embodiments of the present invention are based on theconversion of naturally occurring ribonucleases (e.g., humanribonucleases) into toxic proteins that are used to treat or curediseases, particularly cancer and viral infections. The compositionsalso find use in diagnostic applications (e.g., associated with drugscreening or cancer characterization) and research applications. Theseribonucleases can be used as stand alone reagents or they may beincorporated into general or specific delivery systems such as polymers,dendrimers, liposomes, polymeric nanoparticles, or block copolymermicelles. A feature of these proteins is that they are proteins thathave been engineered to be toxic to the cells to which they aredelivered. This feature provides a toxin conjugate that is lesssusceptible to naturally occurring inhibitors of the toxin. Anotherfeature is that their starting point was preferably a natural protein(e.g., a natural human protein) and not a non-natural (e.g., non-human)protein that had to be modified (e.g., humanized) significantly toescape the immune system. One embodiment of this invention is to combinethese protein toxins with antibodies (e.g., humanized or humanantibodies) for targeting to specific pathogenic cells. These proteinconjugates make it much less likely that when used in vivo, they willinduce side effects or an immune response.

In some preferred embodiments, the present invention provides conjugatesof the EVADE human ribonuclease (Quintessence Biosciences, Madison,Wis.) with a targeting component. As such, the EVADE human ribonucleasesexhibit improved efficacy compared to the amphibian ribonucleasesbecause the specific ribonucleolytic activity is higher and thelikelihood of side effects and inducing a human immune response islower. In addition, binding to the native inhibitor, ribonucleaseinhibitor, is disrupted for the EVADE ribonucleases. By degradingcellular RNA in target cells, the EVADE ribonucleases inhibit thecellular growth of the tumors and also enhances the anti-cancer effectsof conventional therapies, including chemotherapy and radiation. It isalso contemplated that EVADE human ribonucleases are not retained in thehuman kidney, as are amphibian ribonucleases. Renal toxicity of theamphibian ribonucleases is dose limiting in mice and humans. Withconventional chemotherapy, it is often a problem that membrane baseddrug pumps can eliminate the small molecule anti-cancer drugs from thecancerous cell. This requires that higher doses of the toxic drugs beused. Like the amphibian ribonuclease, it is expected that the EVADEribonucleases are able to make these resistant cells susceptible tostandard levels of treatment so that lower doses are effective and sideeffects reduced. In addition, the EVADE ribonucleases are contemplatedto provide benefit when used in combination with radiotherapy or otherconvention interventions.

In some embodiments, the compositions and methods of the presentinvention are used to treat diseased cells, tissues, organs, orpathological conditions and/or disease states in a subject organism(e.g., a mammalian subject including, but not limited to, humans andveterinary animals), or in in vitro and/or ex vivo cells, tissues, andorgans. In this regard, various diseases and pathologies are amenable totreatment or prophylaxis using the present methods and compositions. Anon-limiting exemplary list of these diseases and conditions includes,but is not limited to, breast cancer, prostate cancer, lymphoma, skincancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma,ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer,glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lungcancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma,lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervicalcarcinoma, testicular carcinoma, bladder carcinoma, pancreaticcarcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma,genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma,myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma,endometrial carcinoma, adrenal cortex carcinoma, malignant pancreaticinsulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosisflngoides, malignant hypercalcemia, cervical hyperplasia, leukemia,acute lymphocytic leukemia, chronic lymphocytic leukemia, acutemyelogenous leukemia, chronic myelogenous leukemia, chronic granulocyticleukemia, acute granulocytic leukemia, hairy cell leukemia,neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera,essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma,soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, andretinoblastoma, and the like, T and B cell mediated autoimmune diseases;inflammatory diseases; infections; hyperproliferative diseases; AIDS;degenerative conditions, vascular diseases, and the like. In someembodiments, the cancer cells being treated are metastatic.

In some preferred embodiments, the ribonuclease or ribonucleaseconjugates of the present invention are co-administered with othermedical interventions, either simultaneously or sequentially. Forexample, for cancer therapy, any oncolytic agent that is routinely usedin a cancer therapy may be co-administered with the compositions andmethods of the present invention. For example, the U.S. Food and DrugAdministration maintains a formulary of oncolytic agents approved foruse in the United States. International counterpart agencies to theU.S.F.D.A. maintain similar formularies. Table 4 provides a list ofexemplary antineoplastic agents approved for use in the U.S. Thoseskilled in the art will appreciate that the “product labels” required onall U.S. approved chemotherapeutics describe approved indications,dosing information, toxicity data, and the like, for the exemplaryagents. It is contemplated, that in some cases, co-administration withthe compositions of the present invention permits lower doses of suchcompounds, thereby reducing toxicity. TABLE 4 Aldesleukin PROLEUKINChiron Corp., (des-alanyl-1, serine-125 human Emeryville, CAinterleukin-2) Alemtuzumab CAMPATH Millennium and (IgG1κ anti CD52antibody) ILEX Partners, LP, Cambridge, MA Alitretinoin PANRETIN Ligand(9-cis-retinoic acid) Pharmaceuticals, Inc., San Diego CA AllopurinolZYLOPRIM GlaxoSmithKline, (1,5-dihydro-4 H-pyrazolo[3,4- ResearchTriangle d]pyrimidin-4-one monosodium salt) Park, NC Altretamine HEXALENUS Bioscience, West (N,N,N′,N′,N″,N″,-hexamethyl-1,3,5- Conshohocken, PAtriazine-2,4,6-triamine) Amifostine ETHYOL US Bioscience (ethanethiol,2-[(3-aminopropyl)amino]-, dihydrogen phosphate (ester)) AnastrozoleARIMIDEX AstraZeneca (1,3-Benzenediacetonitrile, a,a,a′,a′-Pharmaceuticals, LP, tetramethyl-5-(1H-1,2,4-triazol-1- Wilmington, DEylmethyl)) Arsenic trioxide TRISENOX Cell Therapeutic, Inc., Seattle, WAAsparaginase ELSPAR Merck & Co., Inc., (L-asparagine amidohydrolase,type EC-2) Whitehouse Station, NJ BCG Live TICE BCG Organon Teknika,(lyophilized preparation of an attenuated Corp., Durham, NC strain ofMycobacterium bovis (Bacillus Calmette-Gukin [BCG], substrain Montreal)bexarotene capsules TARGRETIN Ligand(4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8- Pharmaceuticalspentamethyl-2-napthalenyl) ethenyl] benzoic acid) bexarotene gelTARGRETIN Ligand Pharmaceuticals Bleomycin BLENOXANE Bristol-MyersSquibb (cytotoxic glycopeptide antibiotics Co., NY, NY produced byStreptomyces verticillus; bleomycin A₂ and bleomycin B₂) CapecitabineXELODA Roche (5′-deoxy-5-fluoro-N- [(pentyloxy)carbonyl]-cytidine)Carboplatin PARAPLATIN Bristol-Myers Squibb (platinum, diammine [1,1-cyclobutanedicarboxylato(2-)-0,0′]-,(SP-4- 2)) Carmustine BCNU, BICNUBristol-Myers Squibb (1,3-bis(2-chloroethyl)-1-nitrosourea) Carmustinewith Polifeprosan 20 Implant GLIADEL WAFER Guilford Pharmaceuticals,Inc., Baltimore, MD Celecoxib CELEBREX Searle (as4-[5-(4-methylphenyl)-3- Pharmaceuticals,(trifluoromethyl)-1H-pyrazol-1-yl] England benzenesulfonamide)Chlorambucil LEUKERAN GlaxoSmithKline(4-[bis(2chlorethyl)amino]benzenebutanoic acid) Cisplatin PLATINOLBristol-Myers Squibb (PtCl₂H₆N₂) Cladribine LEUSTATIN, 2-CDA R. W.Johnson (2-chloro-2′-deoxy-b-D-adenosine) Pharmaceutical ResearchInstitute, Raritan, NJ Cyclophosphamide CYTOXAN, NEOSAR Bristol-MyersSquibb (2-[bis(2-chloroethyl)amino] tetrahydro- 2H-13,2-oxazaphosphorine2-oxide monohydrate) Cytarabine CYTOSAR-U Pharmacia & Upjohn(1-b-D-Arabinofuranosylcytosine, Company C₉H₁₃N₃O₅) cytarabine liposomalDEPOCYT Skye Pharmaceuticals, Inc., San Diego, CA Dacarbazine DTIC-DOMEBayer AG, (5-(3,3-dimethyl-1-triazeno)-imidazole-4- Leverkusen,carboxamide (DTIC)) Germany Dactinomycin, actinomycin D COSMEGEN Merck(actinomycin produced by Streptomyces parvullus, C₆₂H₈₆N₁₂O₁₆)Darbepoetin alfa ARANESP Amgen, Inc., (recombinant peptide) ThousandOaks, CA daunorubicin liposomal DANUOXOME Nexstar((85-cis)-8-acetyl-10-[(3-amino-2,3,6- Pharmaceuticals, Inc.,trideoxy-a-L-lyxo-hexopyranosyl)oxy]- Boulder, CO7,8,9,10-tetrahydro-6,8,11-trihydroxy-1- methoxy-5,12-naphthacenedionehydrochloride) Daunorubicin HCl, daunomycin CERUBIDINE Wyeth Ayerst, ((1S,3 S)-3-Acetyl-1,2,3,4,6,11- Madison, NJhexahydro-3,5,12-trihydroxy-10-methoxy- 6,11-dioxo-1-naphthacenyl3-amino-2,3,6- trideoxy-(alpha)-L-lyxo-hexopyranoside hydrochloride)Denileukin diftitox ONTAK Seragen, Inc., (recombinant peptide)Hopkinton, MA Dexrazoxane ZINECARD Pharmacia & Upjohn((S)-4,4′-(1-methyl-1,2-ethanediyl)bis-2,6- Company piperazinedione)Docetaxel TAXOTERE Aventis ((2R,3S)-N-carboxy-3-phenylisoserine, N-Pharmaceuticals, Inc., tert-butyl ester, 13-ester with 5b-20-epoxy-Bridgewater, NJ 12a,4,7b,10b,13a-hexahydroxytax-11-en- 9-one 4-acetate2-beuzoate, trihydrate) Doxorubicin HCl ADRIAMYCIN, Pharmacia & Upjohn(8S,10S)-10-[(3-amino-2,3,6-trideoxy-a-L- RUBEX Companylyxo-hexopyranosyl)oxy]-8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1- methoxy-5,12-naphthacenedionehydrochloride) doxorubicin ADRIAMYCIN PFS Pharmacia & Upjohn INTRAVENOUSCompany INJECTION doxorubicin liposomal DOXIL Sequus Pharmaceuticals,Inc., Menlo park, CA dromostanolone propionate DROMOSTANOLONE Eli Lilly& Company, (17b-Hydroxy-2a-methyl-5a-androstan-3- Indianapolis, IN onepropionate) dromostanolone propionate MASTERONE Syntex, Corp., PaloINJECTION Alto, CA Elliott's B Solution ELLIOTT'S B Orphan Medical, IncSOLUTION Epirubicin ELLENCE Pharmacia & Upjohn((8S-cis)-10-[(3-amino-2,3,6-trideoxy-a-L- Companyarabino-hexopyranosyl)oxy]-7,8,9,10- tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12- naphthacenedione hydrochloride) Epoetinalfa EPOGEN Amgen, Inc (recombinant peptide) Estramustine EMCYTPharmacia & Upjohn (estra-1,3,5(10)-triene-3,17-diol(17(beta))-, Company3-[bis(2-chloroethyl)carbamate] 17- (dihydrogen phosphate), disodiumsalt, monohydrate, or estradiol 3-[bis(2- chloroethyl)carbamate]17-(dihydrogen phosphate), disodium salt, monohydrate) Etoposidephosphate ETOPOPHOS Bristol-Myers Squibb (4′-Demethylepipodophyllotoxin9-[4,6-O- (R)-ethylidene-(beta)-D-glucopyranoside], 4′-(dihydrogenphosphate)) etoposide, VP-16 VEPESID Bristol-Myers Squibb(4′-demethylepipodophyllotoxin 9-[4,6-0-(R)-ethylidene-(beta)-D-glucopyranoside]) Exemestane AROMASIN Pharmacia& Upjohn (6-methylenandrosta-1,4-diene-3,17-dione) Company FilgrastimNEUPOGEN Amgen, Inc (r-metHuG-CSF) floxuridine (intraarterial) FUDRRoche (2′-deoxy-5-fluorouridine) Fludarabine FLUDARA BerlexLaboratories, (fluorinated nucleotide analog of the Inc., Cedar Knolls,antiviral agent vidarabine, 9-b-D- NJ arabinofuranosyladenine (ara-A))Fluorouracil, 5-FU ADRUCIL ICN Pharmaceuticals,(5-fluoro-2,4(1H,3H)-pyrimidinedione) Inc., Humacao, Puerto RicoFulvestrant FASLODEX IPR Pharmaceuticals, (7-alpha-[9-(4,4,5,5,5-pentaGuayama, Puerto fluoropentylsulphinyl) nonyl]estra-1,3,5- Rico(10)-triene-3,17-beta-diol) Gemcitabine GEMZAR Eli Lilly(2′-deoxy-2′,2′-difluorocytidine monohydrochloride (b-isomer))Gemtuzumab Ozogamicin MYLOTARG Wyeth Ayerst (anti-CD33 hP67.6) Goserelinacetate ZOLADEX IMPLANT AstraZeneca (acetate salt of [D- PharmaceuticalsSer(But)⁶,Azgly¹⁰]LHRH; pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro- Azgly-NH2 acetate[C₅₉H₈₄N₁₈O₁₄.(C₂H₄O₂)_(x) Hydroxyurea HYDREA Bristol-Myers SquibbIbritumomab Tiuxetan ZEVALIN Biogen IDEC, Inc., (immunoconjugateresulting from a Cambridge MA thiourea covalent bond between themonoclonal antibody Ibritumomab and the linker-chelator tiuxetan [N-[2-bis(carboxymethyl)amino]-3-(p- isothiocyanatophenyl)-propyl]-[N-[2-bis(carboxymethyl)amino]-2-(methyl)- ethyl]glycine) Idarubicin IDAMYCINPharmacia & Upjohn (5,12-Naphthacenedione, 9-acetyl-7-[(3- Companyamino-2,3,6-trideoxy-(alpha)-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro- 6,9,11-trihydroxyhydrochloride,(7S-cis)) Ifosfamide IFEX Bristol-Myers Squibb (3-(2-chloroethyl)-2-[(2-chloroethyl)amino]tetrahydro-2H-1,3,2- oxazaphosphorine 2-oxide)Imatinib Mesilate GLEEVEC Novartis AG, Basel,(4-[(4-Methyl-1-piperazinyl)methyl]-N-[4- Switzerlandmethyl-3-[[4-(3-pyridinyl)-2- pyrimidinyl]amino]-phenyl]benzamidemethanesulfonate) Interferon alfa-2a ROFERON-A Hoffmann-La Roche,(recombinant peptide) Inc., Nutley, NJ Interferon alfa-2b INTRON ASchering AG, Berlin, (recombinant peptide) (LYOPHILIZED GermanyBETASERON) Irinotecan HCl CAMPTOSAR Pharmacia & Upjohn((4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino) Companycarbonyloxy]-1H-pyrano[3′,4′:6,7] indolizino[1,2-b] quinoline-3,14(4H,12H) dione hydrochloride trihydrate) Letrozole FEMARA Novartis(4,4′-(1H-1,2,4-Triazol-1-ylmethylene) dibenzonitrile) LeucovorinWELLCOVORIN, Immunex, Corp., (L-Glutamic acid, N[4[[(2amino-5-formyl-LEUCOVORIN Seattle, WA 1,4,5,6,7,8 hexahydro4oxo6-pteridinyl)methyl]amino]benzoyl], calcium salt (1:1)) Levamisole HClERGAMISOL Janssen Research ((−)-(S)-2,3,5,6-tetrahydro-6- Foundation,phenylimidazo [2,1-b] thiazole Titusville, NJ monohydrochlorideC₁₁H₁₂N₂S.HCl) Lomustine CEENU Bristol-Myers Squibb(1-(2-chloro-ethyl)-3-cyclohexyl-1- nitrosourea) Meclorethamine,nitrogen mustard MUSTARGEN Merck (2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride) Megestrol acetate MEGACE Bristol-MyersSquibb 17α(acetyloxy)-6-methylpregna-4,6- diene-3,20-dione Melphalan,L-PAM ALKERAN GlaxoSmithKline (4-[bis(2-chloroethyl)amino]-L-phenylalanine) Mercaptopurine, 6-MP PURINETHOL GlaxoSmithKline(1,7-dihydro-6H-purine-6-thione monohydrate) Mesna MESNEX Asta Medica(sodium 2-mercaptoethane sulfonate) Methotrexate METHOTREXATE LederleLaboratories (N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]- L-glutamic acid) MethoxsalenUVADEX Therakos, Inc., Way (9-methoxy-7H-furo[3,2-g][1]-benzopyran-Exton, Pa 7-one) Mitomycin C MUTAMYCIN Bristol-Myers Squibb mitomycin CMITOZYTREX SuperGen, Inc., Dublin, CA Mitotane LYSODREN Bristol-MyersSquibb (1,1-dichloro-2-(o-chlorophenyl)-2-(p- chlorophenyl)ethane)Mitoxantrone NOVANTRONE Immunex (1,4-dihydroxy-5,8-bis[[2-[(2-Corporation hydroxyethyl)amino]ethyl]amino]-9,10- anthracenedionedihydrochloride) Nandrolone phenpropionate DURABOLIN-50 Organon, Inc.,West Orange, NJ Nofetumomab VERLUMA Boehringer Ingelheim Pharma KG,Germany Oprelvekin NEUMEGA Genetics Institute, (IL-11) Inc., Alexandria,VA Oxaliplatin ELOXATIN Sanofi Synthelabo,(cis-[(1R,2R)-1,2-cyclohexanediamine- Inc., NY, NY N,N′][oxalato(2-)-O,O′] platinum) Paclitaxel TAXOL Bristol-Myers Squibb(5β,20-Epoxy-1,2a,4,7β,10β,13a- hexahydroxytax-11-en-9-one4,10-diacetate 2-benzoate 13-ester with (2R, 3S)-N-benzoyl-3-phenylisoserine) Pamidronate AREDIA Novartis (phosphonic acid(3-amino-1- hydroxypropylidene) bis-, disodium salt, pentahydrate,(APD)) Pegademase ADAGEN Enzon ((monomethoxypolyethylene glycol(PEGADEMASE Pharmaceuticals, Inc., succinimidyl) 11-17-adenosine BOVINE)Bridgewater, NJ deaminase) Pegaspargase ONCASPAR Enzon(monomethoxypolyethylene glycol succinimidyl L-asparaginase)Pegfilgrastim NEULASTA Amgen, Inc (covalent conjugate of recombinantmethionyl human G-CSF (Filgrastim) and monomethoxypolyethylene glycol)Pentostatin NIPENT Parke-Davis Pharmaceutical Co., Rockville, MDPipobroman VERCYTE Abbott Laboratories, Abbott Park, IL Plicamycin,Mithramycin MITHRACIN Pfizer, Inc., NY, NY (antibiotic produced byStreptomyces plicatus) Porfimer sodium PHOTOFRIN QLT Phototherapeutics,Inc., Vancouver, Canada Procarbazine MATULANE Sigma Tau(N-isopropyl-μ-(2-methylhydrazino)-p- Pharmaceuticals, Inc., toluamidemonohydrochloride) Gaithersburg, MD Quinacrine ATABRINE Abbott Labs(6-chloro-9-(1-methyl-4-diethyl-amine) butylamino-2-methoxyacridine)Rasburicase ELITEK Sanofi-Synthelabo, (recombinant peptide) Inc.,Rituximab RITUXAN Genentech, Inc., (recombinant anti-CD20 antibody)South San Francisco, CA Sargramostim PROKINE Immunex Corp (recombinantpeptide) Streptozocin ZANOSAR Pharmacia & Upjohn (streptozocin2-deoxy-2- Company [[(methylnitrosoamino)carbonyl]amino]- a(andb)-D-glucopyranose and 220 mg citric acid anhydrous) Talc SCLEROSOLBryan, Corp., (Mg₃Si₄O₁₀(OH)₂) Woburn, MA Tamoxifen NOLVADEX AstraZeneca((Z)2-[4-(1,2-diphenyl-1-butenyl) Pharmaceuticalsphenoxy]-N,N-dimethylethanamine 2- hydroxy-1,2,3-propanetricarboxylate(1:1)) Temozolomide TEMODAR Schering(3,4-dihydro-3-methyl-4-oxoimidazo[5,1- d]-as-tetrazine-8-carboxamide)teniposide, VM-26 VUMON Bristol-Myers Squibb(4′-demethylepipodophyllotoxin 9-[4,6-0- (R)-2-thenylidene-(beta)-D-glucopyranoside]) Testolactone TESLAC Bristol-Myers Squibb(13-hydroxy-3-oxo-13,17-secoandrosta-1,4- dien-17-oic acid[dgr]-lactone) Thioguanine, 6-TG THIOGUANINE GlaxoSmithKline(2-amino-1,7-dihydro-6H-purine-6- thione) Thiotepa THIOPLEX Immunex(Aziridine, 1,1′,1″- Corporation phosphinothioylidynetris-, or Tris (1-aziridinyl) phosphine sulfide) Topotecan HCl HYCAMTTN GlaxoSmithKline((S)-10-[(dimethylamino) methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7] indolizino [1,2-b] quinoline-3,14-(4H,12H)-dione monohydrochloride) Toremifene FARESTON Roberts(2-(p-[(Z)-4-chloro-1,2-diphenyl-1- Pharmaceuticalbutenyl]-phenoxy)-N,N- Corp., Eatontown, NJ dimethylethylamine citrate(1:1)) Tositumomab, I 131 Tositumomab BEXXAR Corixa Corp., Seattle,(recombinant murine immunotherapeutic WA monoclonal IgG_(2a) lambdaanti-CD20 antibody (I 131 is a radioimmunotherapeutic antibody))Trastuzumab HERCEPTIN Genentech, Inc (recombinant monoclonal IgG₁ kappaanti- HER2 antibody) Tretinoin, ATRA VESANOID Roche (all-trans retinoicacid) Uracil Mustard URACIL MUSTARD Roberts Labs CAPSULES Valrubicin,N-trifluoroacetyladriamycin- VALSTAR Anthra --> Medeva 14-valerate((2S-cis)-2-[1,2,3,4,6,11-hexahydro- 2,5,12-trihydroxy-7methoxy-6,11-dioxo- [[4 2,3,6-trideoxy-3-[(trifluoroacetyl)-amino-α-L-lyxo-hexopyranosyl]oxyl]-2- naphthacenyl]-2-oxoethylpentanoate) Vinblastine, Leurocristine VELBAN Eli Lilly(C₄₆H₅₆N₄O₁₀.H₂SO₄) Vincristine ONCOVLIN Eli Lilly (C₄₆H₅₆N₄O₁₀.H₂SO₄)Vinorelbine NAVELBINE GlaxoSmithKline (3′,4′-didehydro-4′-deoxy-C′-norvincaleukoblastine [R-(R*,R*)-2,3- dihydroxybutanedioate(1:2)(salt)]) Zoledronate, Zoledronic acid ZOMETA Novartis((1-Hydroxy-2-imidazol-1-yl- phosphonoethyl) phosphonic acidmonohydrate)

A current and still developing approach to cancer therapy involves usingcancer cell-specific reagents to target a malignant tumor. These toxicreagents can be produced by attaching a toxic payload to a cell-specificdelivery vector. Over the past few years, a wide variety oftumor-specific targeting proteins, including antibodies, antibodyfragments, and ligands for cell surface receptors have been developedand clinically tested. These targeting molecules have been conjugated toseveral classes of therapeutic toxins such as small molecule drugs,enzymes, radioisotopes, protein toxins, and other toxins for specificdelivery to patients. While these efforts have made meaningful inroadsto treat cancers, significant challenges lie ahead to develop moreeffective toxins, to create more robust and specific delivery systems,and to design therapeutic proteins and protein vectors that evade immunesurveillance in humans.

Ribonuclease (RNase) proteins have been tested as human therapeuticsbecause they selectively target tumor cells; this has been demonstratedmost clearly with an RNase from Rana pipiens early embryos. Rana pipiensis a species of leopard frogs and its embryonic RNase is distantlyrelated to the more highly conserved bovine and human pancreaticribonucleases. In mammalian cells, pancreatic-type ribonucleases, suchas RNase A, are secretory enzymes that catalyze the degradation of RNAto ribonucleotides and their activity is inhibited by binding toribonuclease inhibitor (RI), a ubiquitous cytosolic protein.Ribonuclease inhibitor binds exceptionally tight to pancreatic-typeRNases, abating their activity and thereby making them non-toxic tonormal or cancer cells. If the RNase activity is inhibited, the cellularRNA is undamaged and the cell remains viable. In normal cells theribonuclease activity is tightly controlled, but if ribonucleaseactivity is uncontrolled, the ribonucleolytic activity destroys cellularRNA and kills the cell. There are two main approaches to making aribonuclease toxic to human cells, especially cancer cells. The firstapproach is to select a ribonuclease that is evolutionarily distant tohumans and is not inhibited by human ribonuclease inhibitor protein. Thefrog (Rana pipiens) ribonuclease, when placed in a human cell, is notstrongly inhibited by RI and its RNase activity destroys cellular RNAand kills the target cell. This has been the approach with a specificRana pipiens RNase called Ranpimase. Ranpimase is generic name of thepharmaceutical that is described and claimed in U.S. Pat. No. 5,559,212and that is presently known by the registered trademark ONCONASE.

The second approach is to mutate mammalian ribonucleases so that theymaintain high levels of ribonucleolytic activity but are notsignificantly inhibited by human ribonuclease inhibitor. These mutatedenzymes provide high levels of ribonucleolytic activity within cancercells because of disruption of binding to RI. This unregulated activityis particularly lethal to cancer cells. This mutation approach has beendemonstrated with the mammalian proteins bovine RNase A and human RNaseI and is described in U.S. Pat. Nos. 5,389,537 and 6,280,991, thedisclosures of which are herein incorporated by reference in theirentireties.

An ideal protein candidate for cancer therapy would be more toxic totumor cells compared to non-cancerous cells and would be targetable to aspecific tumor. This candidate should have few side effects and shouldnot stimulate a human immune response. Therapeutic proteins that elicitimmune responses in humans are always problematic and oftenunacceptable. The present invention provides ribonuclease conjugatesthat are derived from mutated RNases that exhibit low immunogenicity andside effects and still maintain high ribonucleolytic activity resultingin cancer-specific toxicity.

Certain preferred embodiments of the present invention are describedbelow. While these embodiments are illustrated with variant humanribonuclease proteins and antibody targeting moieties, the presentinvention is not so limited.

Therapeutic Antibodies and Delivery of Cytotoxins

Antibodies are glycoprotein molecules produced by white blood cells(B-lymphocytes) of the immune system and their function is to recognizeand bind to matter harmful to the organism. Once an antigen is marked byan antibody, it is destroyed by other components of the immune system. Atypical organism makes millions of different antibodies, each designedto bind a specific epitope (or antigenic determinant) on the foreignantigen. Antibodies naturally combine specificity (the ability toexquisitely discriminate diverse harmful molecules) and affinity (theability to tightly lock onto those targets) with the ability to recruiteffector functions of the immune system such as antibody- andcomplement-mediated cytolysis and antibody-dependent cell-mediatedcytotoxicity (ADCC). Many new therapeutic approaches involvingantibodies have succeeded in potentiating the natural antibody functionsto treat or cure diseases.

Alternatively, a “toxic payload” (such as a radioactive element or atoxin) attached to the antibody can be accurately delivered to thepathogenic target. The following table lists the mechanisms of somecancer therapeutic antibodies, including three antibody conjugates thatcarry a toxic payload for lymphomas and leukemias. (Drug DiscoveryToday, Vol. 8, No. 11 June 2003). Two of the conjugates, ZEVALIN andBEXXAR, carry radioactive iodine as the toxin and the third, MYLOTARG,carries a cytotoxic antitumor antibiotic, calicheaminin which isisolated from a bacterial fermentation. The Mylotarg antibody bindsspecifically to the CD33 antigen which is expressed on the surface ofleukemic blasts that are found in more than 80% of patients with acutemyeloid leukemia (AML). The antibody in this conjugate has approximately98.3% of its amino acid sequences derived from human origins. TABLE 2Antibody Mode of Action Product Antibody Target Blockade Ligand bindingERBITUX EGF receptor HUMAX-EGFR EGF receptor Complement DependentCytotoxicity RITUXAN CD20 HUMAX-CD20 CD20 CAMPATH-1H CD52 Antibodydependent cell-mediated RIXTUXAN CD20 cytotoxicity HUMAX-CD20 CD20HERCEPTIN Her-2/neu HUMAX-EGFR EGF receptor Apoptosis induction VariousIdiotypeB cell tumors Disruption signaling 2C4 Her-2/neu (PERTUZUMAB)Inhibition angiogenesis AVASTIN VEGF Targeted radiolysis conjugateZEVALIN CD20 BEXXAR CD20 Toxin-mediated killing by conjugate MYLOTARGCD33 Antagonist activity MDX-010 CTLA4 Agonist activity Various CD40,CD137 Antagonist activity Preclinical MAb Epithelial cell receptorprotein tyrosine kinase (EphA2) Antagonist activity Phase II MAb alpha 5beta 3 integrin (receptor) Antagonist activity Phase I bispecificCD19/CD3 single chain monoclonal antibody Antagonist activityPreclinical MAb Interleukin 9 Antagonist activity RespiGam Respiratorysyncytial virus Polyclonal Antibody Antagonist activity Phase II MAb CD2Catalytic Activity MAb Cocaine cleavage Anti-infective, bacteria MAbbacteria Immunosuppressive Agents MAb Graft versus Host DiseaseAnti-infective, virus MAb Human metapneumovirus Cytostatic agent MAbPlatelet derived growth factor Cancer growth and metastosis PreclinicalMAb Human beta hydroxylases Treatment of autoimmune disease MAb Medi 507Mixed lymphocyte responses Anti-infective, virus Polyclonal antibodycytomegalovirus Anti-idiotype antibody MAb Neu-glycolyl-GM3 gangliosideProdrug carrier MAb Immungen's CC 1065 prodrugs Toxin-mediated killingby conjugate Preclinical MAb Various by Immunogen and taxane derivativesToxin-mediated killing by conjugate Cantuzumab Can Ag receptor bymertansine immunogen conjugate Toxin-mediated killing by conjugate PhaseII MAb CD56 maytansinoid conjugate Toxin for mitosis inhibition MAbmaitansine various conjugate Toxin-mediated killing by conjugatePreclinical MAb Antigen on squamous cell cytotoxic drug cancer(Immunogen) DM1 conjugate

Any of the targeting antibodies or agents used in these products mayalso be employed by the compositions and methods of the presentinvention.

Generally, the most specific method for targeting toxins is the use ofmonoclonal antibodies or antibody fragments that are designed torecognize surface antigens specific to tumor cells. Because normal cellslack the surface antigens, they are not targeted and killed by the toxinconjugate. Whole antibodies have two domains: a variable domain thatgives the antibody its affinity and binding specificity and a constantdomain that interacts with other portions of the immune system tostimulate immune responses in the host organism. The variable domain iscomposed of the complementarity determining regions (CDRs), which bindto the antibody's target, and a framework region that anchors the CDRsto the rest of the antibody and helps maintain CDR shape. The six CDR'sin each antibody differ in length and sequence between differentantibodies and are mainly responsible for the specificity (recognition)and affinity (binding) of the antibodies to their target markers.

The functions of antibodies are reflected in their characteristicthree-dimensional structure, which is ultimately determined by theprimary sequence of amino acids and how those amino acids fold into afunctional 3-dimensional protein chain. A step in developing therapeuticmonoclonal antibodies is to simultaneously optimize biochemical andcellular functions for anti-cancer performance and still keep theprotein as humanlike as possible to minimize any anti-antibody humanimmune response.

Monoclonal antibodies were originally produced in mice, but when theyare used in human therapeutic applications, they present formidableobstacles. Mouse antibodies are recognized as foreign by the humanimmune system and thus they provoke the Human Anti-Mouse Antibody orHAMA reaction. The HAMA reaction alters the mouse monoclonaleffectiveness and can cause severe adverse symptoms in the recipient.Furthermore, mouse antibodies are simply not as effective as humanantibodies in mediating the human immune system to destroy the malignantcells. For these reasons, it is often desired to design monoclonalantibodies that are as humanlike as possible but still maintain optimalbiochemical, immunological, and therapeutic performance.

There are several factors that influence whether a therapeutic antibodywill induce an immune response in the human host. These include theefficiency of uptake by an APC (antigen presenting cell) viapinocytosis, receptor-mediated endocytosis, or phagocytosis. Theefficiency of uptake is in turn influenced by the route ofadministration, the solubility (or aggregation) of the protein, itsreceptor binding specificity, and whether the protein is recognized byclass II major histocompatibility complex (MHC) molecules, T-cellreceptors (TCR), and B-cell receptors (BCR). One of the moststraightforward ways to evade the human immune response is to make thetherapeutic protein sequence and structure as humanlike as possible.

Two main approaches have emerged to produce human or humanizedtherapeutic monoclonal antibodies, either used alone as a therapeutic oras a carrier for a toxin. These include 1) ‘humanizing’ mouse or othernon-human antibodies to make them compatible with the human immunesystem and 2) producing fully human antibodies in transgenic mice or byusing genetic engineering methods in the laboratory. The processes haveproduced several categories of monoclonal antibodies. These includemouse, chimaeric, humanized and human antibodies. They are describedbriefly below:

-   -   1. Murine Monoclonal antibodies from mice and rats: The original        Kohler and Milstein technology from 1975 provided mouse        monoclonal antibodies using a hybridoma technology. These have        been used therapeutically. In 1986, the first approved use of        mouse monoclonals was for transplant patients whose immune        system was suppressed to avoid organ rejection. Rodent        antibodies tend to provoke strong Human anti-Murine Antibody        (HAMA) immune responses that restrict their usefulness for        repeated application in the same patient.    -   2. Chimaeric Antibodies: These are mutated antibodies in which        the entire variable regions of a functional mouse antibody are        joined to human constant regions. These antibodies have human        effector functions from the constant (Fc regions) such as        activating complement and recruiting immune cells. These        chimaeric antibodies also reduce the immunogenicity (HAMA)        caused by the mouse constant region.    -   3. Humanized/CDR grafted/Reshaped antibodies: These antibodies        are more humanlike than chimaeric antibodies because only the        complementarity determining regions from the mouse antibody        variable regions are combined with framework regions from human        variable regions. Because these antibodies are more human-like        than chimaeric antibodies, it is expected they could be designed        to be less immunogenic when given to human in recurring        therapeutic doses. Using computer modeling software to guide the        humanization of murine antibodies or random shuffling of        sequences followed by screening, it is possible to design an        antibody that retains most or all of the binding affinity and        specificity of the murine antibody but which is >90% human.    -   4. Human antibodies from immune donors: Some antibodies have        been rescued from immune human donors using either Epstein Barr        Virus transformation of B-cells or by PCR cloning and phage        display. By definition these antibodies are completely human in        origin.    -   5. Fully human antibodies from phage libraries: Synthetic phage        libraries have been created which use randomized combinations of        synthetic human antibody V-regions. By panning these libraries        against a target antigen, these so called ‘fully human        antibodies’ are assumed to be very human but possibly more        diverse than natural antibodies.    -   6. Fully human antibodies from transgenic mice: Transgenic mice        have been created that have functional human immunoglobulin        germline genes sequences. These transgenic mice produce        human-like antibodies when immunized.

The human antibodies produced by methods 4, 5, and 6 are typically mostdesired because they produce a starting antibody that contains no mouseor otherwise “foreign” protein sequences that should stimulate an immuneresponse in human patient. This approach (in 4, 5, and 6) also canbypass the challenge of substituting mouse CDR regions into humanframeworks that often alters the 3-dimensional structure of the variableregion, thereby changing the antibody's binding and specificity. Thisapproach (in 4, 5, 6) successfully produced an anti-CD3 antibody. Themurine version elicited neutralizing antibodies after a single dose inall patients tested, while a humanized version was only immunogenic in25% of patients following multiple injections.

Besides making monoclonal antibodies as human-like as possible in theprimary sequence to escape the human immune response, several otherapproaches make antibodies less immunogenic and more therapeuticallyeffective are available. One approach is to covalently modify theantibody surface with reagents such as polyethylene glycol (PEG) tosuppress its antigenicity and improve its solubility. These biochemicalmodifications also can have several other benefits such as reducedtoxicity, increased bioavailability, and improved efficacy. Anotherapproach is to use antibody fragments in which the potentially antigenicparts of the mouse antibody, such as the constant region, have beenremoved. This approach typically works only when the regulatorycomponents within the antibody constant region are not required fortherapeutic efficacy. Neither of these approaches has proven completelysatisfactory, which has driven the humanization effort to produce ‘theideal’ antibody candidate mentioned above.

In addition to antibody delivery vectors, toxic molecules can bedelivered to cancer cells using several other specific and non-specificvectors including peptides, polymers, dendrimers, liposomes, polymericnanoparticles, and block copolymer micelles. For example, peptides thatbind to the leutinizing hormone-releasing hormone have been used totarget a small molecule toxin, camptothecin, to ovarian cancer cells(Journal of Controlled Release, 2003, 91, 61-73.).

Ribonucleases that evade ribonuclease inhibitor protein are effectivetoxins in human cells, particularly against cancer cells. The followingreferences, each of which is herein incorporated by reference in itsentirety, describe some chemical conjugates of ribonucleases totargeting proteins (including proteins and antibodies): Newton et al.(2001), Blood 97(2): 528-35, Hursey et al. (2002) Leuk Lymphoma 43(5):953-9, Rybak et al., (1991) Journal of Biological Chemistry 266(31):21202-7, Newton et al. (1992) Journal of Biological Chemistry 267(27):19572-8, Jinno and Ueda (1996) Cancer Chemother Pharmacol 38: 303-308,Yamamura et al. (2002) Eur J Surg 168: 49-54, Jinno et al. (1996) LifeSci 58: 1901-1908, Suzuki et al. (1999) Nature Biotechnology 17(3):265-70, Rybak et al. (1992), Cell Biophys 21(1-3): 121-38, Jinno et al.(2002) Anticancer Res. 22: 4141-4146.

Non-Natural Ribonuclease Polynucleotides

As described above, a new family of non-natural ribonuclease proteinsthat have been discovered. This family was identified bystructure-function analys for ribonuclease sequence with desiredcytotoxic activities. Accordingly, the present invention providesnucleic acids encoding these novel non-natural ribonucleases, homologs,and variants (e.g., mutations and polyporphisms). In some embodiments,the present invention provides polynucleotide sequences encoding any ofthe amino acid sequences listed in Tables 1-3. The present inventionalso provides nucleic acid that are capable of hybridizing to suchnucleic acid sequences under conditions of low to high stringency aslong as the polynucleotide sequence capable of hybridizing encodes aprotein that retains a biological activity of a ribonuclease (e.g.,cytotoxic activity). The above nucleic acid molecules may also beassociated with coding sequences of targeting molecules (e.g.,antibodies) such that the produced amino acid sequence is a fusionbetween the ribonuclease and the targeting molecule.

In still other embodiments of the present invention, the nucleotidesequences of the present invention may be engineered in order to alter aribonuclease coding sequence for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing and/orexpression of the gene product. For example, mutations may be introducedusing techniques that are well known in the art (e.g., site-directedmutagenesis to insert new restriction sites, to change codon preference,etc.).

It is contemplated that it is possible to modify the structure of apeptide having a function (e.g., ribonuclease function) for suchpurposes as increasing activity of the ribonuclease (e.g., cytotoxicactivity). Such modified peptides are considered functional equivalentsof peptides having an activity of a ribonuclease as defined herein. Amodified peptide can be produced in which the nucleotide sequenceencoding the polypeptide has been altered, such as by substitution,deletion, or addition. In particularly preferred embodiments, thesemodifications do not significantly reduce the cytotoxic activity of themodified ribonuclease. In other words, construct “X” can be evaluated inorder to determine whether it is a member of the genus of modified orvariant ribonucleases of the present invention as defined functionally,rather than structurally. In preferred embodiments, the activity of avariant ribonuclease is evaluated by any known screening method,including those described herein expressly or by reference.

Moreover, variant forms of ribonucleases, as shown in Tables 2 and 3,are provided. Further variations of these compositions are contemplated,including structural and functional equivalents. For example, it iscontemplated that isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid (i.e., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Accordingly, someembodiments of the present invention provide variants of ribonucleasesdisclosed herein containing conservative replacements. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains. Genetically encoded amino acidscan be divided into four families: (1) acidic (aspartate, glutamate);(2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan);and (4) uncharged polar (glycine, asparagine, glutamine, cysteine,serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosineare sometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3)aliphatic (glycine, alanine, valine, leucine, isoleucine, serine,threonine), with serine and threonine optionally be grouped separatelyas aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,Biochemistry, pg. 17-21, 2^(nd) ed, WH Freeman and Co., 1981). Whether achange in the amino acid sequence of a peptide results in a functionalhomolog can be readily determined by assessing the ability of thevariant peptide to function in a fashion similar to the referenceprotein. Peptides having more than one replacement can readily be testedin the same manner.

More rarely, a variant includes “nonconservative” changes (e.g.,replacement of a glycine with a tryptophan). Analogous minor variationscan also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues can be substituted, inserted,or deleted without abolishing biological activity can be found usingcomputer programs (e.g., LASERGENE software, DNASTAR Inc., Madison,Wis.).

As described in more detail below, variants may be produced by methodssuch as directed evolution or other techniques for producingcombinatorial libraries of variants. In still other embodiments of thepresent invention, the nucleotide sequences of the present invention maybe engineered in order to alter a ribonuclease coding sequenceincluding, but not limited to, alterations that modify the cloning,processing, localization, secretion, and/or expression of the geneproduct.

Non-Natural Ribonuclease Polypeptides

Non-natural ribonuclease polypeptides are described in Tables 1-3. Thepresent invention also provides fragments, fusion proteins or functionalequivalents of these ribonuclease proteins.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. In some embodiments of the presentinvention, vectors include, but are not limited to, chromosomal,nonchromosomal and synthetic DNA sequences (e.g., derivatives of SV40,bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectorsderived from combinations of plasmids and phage DNA, and viral DNA suchas vaccinia, adenovirus, fowl pox virus, and pseudorabies). It iscontemplated that any vector may be used as long as it is replicable andviable in the host.

In particular, some embodiments of the present invention providerecombinant constructs comprising one or more of the sequences asbroadly described above. In some embodiments of the present invention,the constructs comprise a vector, such as a plasmid or viral vector,into which a sequence of the invention has been inserted, in a forwardor reverse orientation. In still other embodiments, the heterologousstructural sequence is assembled in appropriate phase with translationinitiation and termination sequences. In preferred embodiments of thepresent invention, the appropriate DNA sequence is inserted into thevector using any of a variety of procedures. In general, the DNAsequence is inserted into an appropriate restriction endonucleasesite(s) by procedures known in the art.

Large numbers of suitable vectors are known to those of skill in theart, and are commercially available. Such vectors include, but are notlimited to, the following vectors: 1) Bacterial—pQE70, pQE60, pQE-9(Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia); and 2) Eukaryotic—pWLNEO, pSV2CAT, pOG44,PXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). Any otherplasmid or vector may be used as long as they are replicable and viablein the host. In some preferred embodiments of the present invention,mammalian expression vectors comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation sites, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking non-transcribedsequences. In other embodiments, DNA sequences derived from the SV40splice, and polyadenylation sites may be used to provide the requirednon-transcribed genetic elements.

In certain embodiments of the present invention, the DNA sequence in theexpression vector is operatively linked to an appropriate expressioncontrol sequence(s) (promoter) to direct mRNA synthesis. Promotersuseful in the present invention include, but are not limited to, the LTRor SV40 promoter, the E. coli lac or trp, the phage lambda PL and PR, T3and T7 promoters, and the cytomegalovirus (CMV) immediate early, herpessimplex virus (HSV) thymidine kinase, and mouse metallothionein-Ipromoters and other promoters known to control expression of gene inprokaryotic or eukaryotic cells or their viruses. In other embodimentsof the present invention, recombinant expression vectors include originsof replication and selectable markers permitting transformation of thehost cell (e.g., dihydrofolate reductase or neomycin resistance foreukaryotic cell culture, or tetracycline or ampicillin resistance in E.coli).

In some embodiments of the present invention, transcription of the DNAencoding the polypeptides of the present invention by higher eukaryotesis increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300bp that act on a promoter to increase its transcription. Enhancersuseful in the present invention include, but are not limited to, theSV40 enhancer on the late side of the replication origin bp 100 to 270,a cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

In other embodiments, the expression vector also contains aribosome-binding site for translation initiation and a transcriptionterminator. In still other embodiments of the present invention, thevector may also include appropriate sequences for amplifying expression.

In a further embodiment, the present invention provides host cellscontaining the above-described constructs. In some embodiments of thepresent invention, the host cell is a higher eukaryotic cell (e.g., amammalian or insect cell). In other embodiments of the presentinvention, the host cell is a lower eukaryotic cell (e.g., a yeastcell). In still other embodiments of the present invention, the hostcell can be a prokaryotic cell (e.g., a bacterial cell). Specificexamples of host cells include, but are not limited to, Escherichiacoli, Salmonella typhimurium, Bacillus subtilis, and various specieswithin the genera Pseudomonas, Streptomyces, and Staphylococcus, as wellas Saccharomycees cerivisiae, Schizosaccharomycees pombe, Drosophila S2cells, Spodoptera Sf9 cells, Chinese hamster ovary (CHO) cells, COS-7lines of monkey kidney fibroblasts, (Gluzman, Cell 23:175 [1981]), C127, 3T3, 293, 293T, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. In someembodiments, introduction of the construct into the host cell can beaccomplished by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (See e.g., Davis et al., Basic Methodsin Molecular Biology, [1986]). Alternatively, in some embodiments of thepresent invention, the polypeptides of the invention can besynthetically produced by conventional peptide synthesizers.

Proteins can be expressed in mammalian cells, yeast, bacteria, or othercells under the control of appropriate promoters. Cell-free translationsystems can also be employed to produce such proteins using RNAs derivedfrom the DNA constructs of the present invention. Appropriate cloningand expression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989).

In some embodiments of the present invention, following transformationof a suitable host strain and growth of the host strain to anappropriate cell density, the selected promoter is induced byappropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. In other embodiments of thepresent invention, cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification. In still other embodiments of thepresent invention, microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents.

The polypeptides of the present invention may also be chemicallysynthesized (Gutte, B. and Merrifield, R. B. The synthesis ofribonuclease A. J. Biol. Chem. 1971, 246I, 1722-1741.).

The present invention further contemplates methods of generating sets ofcombinatorial mutants of the present ribonuclease proteins andribonuclease conjugates. Library are screened to generate, for example,novel ribonuclease or ribonulcease conjugate variants with improvedproperties (e.g., cytotoxicity against target cells, cell targeting, lowsystemic toxicity, stability, clearance, and improved storage, handling,and administration).

In some embodiments of the combinatorial mutagenesis approach of thepresent invention, the amino acid sequences for a population ofribonuclease variants or other related proteins are aligned, preferablyto promote the highest homology possible. Such a population of variantscan include, for example, ribonuclease homologs from one or morespecies, or ribonuclease variants from the same species but which differdue to mutation. Amino acids that appear at each position of the alignedsequences are selected to create a degenerate set of combinatorialsequences.

In a preferred embodiment of the present invention, the combinatorialribonuclease library is produced by way of a degenerate library of genesencoding a library of polypeptides which each include at least a portionof potential ribonuclease protein sequences. For example, a mixture ofsynthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential ribonucleasesequences are expressible as individual polypeptides, or alternatively,as a set of larger fusion proteins (e.g., for phage display) containingthe set of ribonuclease sequences therein.

There are many ways by which the library of potential ribonucleasehomologs and variants can be generated from a degenerate oligonucleotidesequence. In some embodiments, chemical synthesis of a degenerate genesequence is carried out in an automatic DNA synthesizer, and thesynthetic genes are ligated into an appropriate gene for expression. Thepurpose of a degenerate set of genes is to provide, in one mixture, allof the sequences encoding the desired set of potential ribonucleasesequences. The synthesis of degenerate oligonucleotides is well known inthe art (See e.g., Narang, Tetrahedron Lett., 39:39 [1983]; Itakura etal., Recombinant DNA, in Walton (ed.), Proceedings of the 3rd ClevelandSymposium on Macromolecules, Elsevier, Amsterdam, pp 273-289 [1981];Itakura et al., Annu. Rev. Biochem., 53:323 [1984]; Itakura et al.,Science 198:1056 [1984]; Ike et al., Nucl. Acid Res., 11:477 [1983]).Such techniques have been employed in the directed evolution of otherproteins (See e.g., Scott et al., Science 249:386-390 [1980]; Roberts etal., Proc. Natl. Acad. Sci. USA 89:2429-2433 [1992]; Devlin et al.,Science 249: 404-406 [1990]; Cwirla et al., Proc. Natl. Acad. Sci. USA87: 6378-6382 [1990]; as well as U.S. Pat. Nos. 5,223,409, 5,198,346,and 5,096,815, each of which is incorporated herein by reference).

It is contemplated that the ribonuclease nucleic acids can be utilizedas starting nucleic acids for directed evolution. These techniques canbe utilized to develop ribonuclease variants having desirableproperties.

In some embodiments, artificial evolution is performed by randommutagenesis (e.g., by utilizing error-prone PCR to introduce randommutations into a given coding sequence). This method requires that thefrequency of mutation be finely tuned. As a general rule, beneficialmutations are rare, while deleterious mutations are common. This isbecause the combination of a deleterious mutation and a beneficialmutation often results in an inactive enzyme. The ideal number of basesubstitutions for targeted gene is usually between 1.5 and 5 (Moore andArnold, Nat. Biotech., 14, 458-67 [1996]; Leung et al., Technique,1:11-15 [1989]; Eckert and Kunkel, PCR Methods Appl., 1: 17-24 [1991];Caldwell and Joyce, PCR Methods Appl., 2:28-33 (1992); and Zhao andArnold, Nuc. Acids. Res., 25:1307-08 [1997]). After mutagenesis, theresulting clones are selected for desirable activity (e.g., screened forribonuclease activity and/or cytotoxicity). Successive rounds ofmutagenesis and selection are often necessary to develop enzymes withdesirable properties. It should be noted that, preferably, only theuseful mutations are carried over to the next round of mutagenesis.

In other embodiments of the present invention, the polynucleotides ofthe present invention are used in gene shuffling or sexual PCRprocedures (e.g., Smith, Nature, 370:324-25 [1994]; U.S. Pat. Nos.5,837,458; 5,830,721; 5,811,238; 5,733,731; all of which are hereinincorporated by reference). Gene shuffling involves random fragmentationof several mutant DNAs followed by their reassembly by PCR into fulllength molecules. Examples of various gene shuffling procedures include,but are not limited to, assembly following DNase treatment, thestaggered extension process (STEP), and random priming in vitrorecombination. In the DNase mediated method, DNA segments isolated froma pool of positive mutants are cleaved into random fragments with DNaseIand subjected to multiple rounds of PCR with no added primer. Thelengths of random fragments approach that of the uncleaved segment asthe PCR cycles proceed, resulting in mutations in present in differentclones becoming mixed and accumulating in some of the resultingsequences. Multiple cycles of selection and shuffling have led to thefunctional enhancement of several enzymes (Stemmer, Nature, 370:398-91[1994]; Stemmer, Proc. Natl. Acad. Sci. USA, 91, 10747-51 [1994];Crameri et al., Nat. Biotech., 14:315-19 [1996]; Zhang et al., Proc.Natl. Acad. Sci. USA, 94:4504-09 [1997]; and Crameri et al., Nat.Biotech., 15:436-38 [1997]).

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations, and forscreening cDNA libraries for gene products having a certain property.Such techniques will be generally adaptable for rapid screening of thegene libraries generated by the combinatorial mutagenesis orrecombination of ribonuclease homologs. The most widely used techniquesfor screening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected.

EXAMPLES Example 1 Exemplary Embodiments

The following Example describes a number of exemplary embodiments of thecompositions and methods of the present invention.

Ribonuclease:

The EVADE family of ribonucleases comprises several members, based onhuman ribonuclease one (RNase I, also known as human pancreaticribonuclease, hpRNase or hRNase). Some of the EVADE ribonucleases havebeen assigned the following numbers (QBI-#####) and the single lettersand numbers describe the amino acid changes. For example, N88C refers toa substitution of Cysteine (C) for Asparagine (N) at position 88.

Amino acid sequence for bovine ribonuclease A 1 10 20 30 40 KETAAAKFERQHMDSSTSA ASSSNYCNQM MKSRNLTKDR CKPVNTFVHE 50 60 70 80 90 SLADVQAVCSQKNVACKNGQ TNCYQSYSTM SITDCRETGS SKYPNCAYKT 100 110 120 TQANKHIIVACEGNPYVPVH FDASV

Amino acid sequence for human pancreatic ribonuclease I 1 10 20 30 40KESRAKKFQ RQHMDSDSSP SSSSIYCNQM MRRRNMIQGR CKPVNIFVHE 50 60 70 80 90PLVDVQNVCF QEKVICKNGQ GNCYKSNSSM HIIDCRLTNG SRYPNCAYRT 100 110 120SPKERHIIVA CEGSPYVPVH FDASVEDST Amino Acid Three Letter AbbreviationsSingle Letter Abbreviations Alanine Ala A Arginine Arg R Asparagine AsnN Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu EGlycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine LysK Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser SThreonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V QBI-50101N88C RNase I QBI-50109 L86E, N88R, G89D, R91D RNase I QBI-50110 R4C,L86E, N88R, G89D, R91D, V118C RNase I QBI-50111 L86E, N88C, R91D RNase IQBI-50112 R4C, L86E, N88C, R91D, V118C RNase I QBI-50118 R4C, N88C,V118C RNase I QBI-50125 K7A, L86E, N88C, R91D RNase I QBI-50126 K7A,L86E, N88R, G89D, R91D RNase I QBI-50127 R4C, K7A, L86E, N88C, R91D,V118C RNase I QBI-50128 R4C, K7A, L86E, N88R, G89D, R91D, VL18C RNase I

In some embodiments, EVADE ribonucleases are conjugated to moleculesthat accelerate their targeting to and uptake by diseased cells (e.g.,cancer, viruses, autoimmune diseases). This type of modification extendsthe utility and enhances the efficacy of the EVADE ribonucleases. AnEVADE ribonuclease that has been modified to carry a Cys at any aminoacid can be readily adapted for use in this conjugation strategy. Aminoacids in the loop region corresponding to amino acids 84-95 of bovineribonuclease A are of particular interest for conjugation. A preferredconjugation partner is an antibody that binds to a cell-specific epitope(e.g. a cancer marker). The antibody is cross-linked to the EVADEribonuclease via a non-cleavable or a cleavable cross-linker. Anon-cleavable chemical cross-linker may includem-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Alternatively, acleavable linker may be used, with the cleavage occurring as a result ofenzymatic activity (e.g., protease or a lactamase) or a change inenvironment (e.g., reducing or acidic environment). A number ofchemistries are available for conjugation to an antibody, including analdehyde (generated by oxidation of carbohydrate), an amine (present onthe lysine side chains), or a thiol (particularly useful for conjugationto Fab′ fragment or scFv). Ribonuclease Antibody Cross-linker categoryExamples thiol of Cys of thiol of Fab bifunctional thiol BMB (8.0 Å)EVADE ribonuclease fragment BMDP (10.2 Å) BMOE (10.9 Å) BM(PEO)₃ (14.7Å) BM(PEO)₄ (17.8 Å) thiol of Cys of non-native Cys of bifunctionalthiol See above EVADE ribonuclease single chain (scFv) thiol of Cys ofThiol of mAb bifunctional thiol See above EVADE ribonuclease (introducedby reaction of small molecule with amines of mAb; e.g withiminothiolane) thiol of Cys of Amine of mAb thiol, amine sulfo-GMBS (6.8Å) EVADE ribonuclease heterobifunctional sulfo-SIAB (10.6 Å) sulfo-EMCS(9.4 Å) thiol of Cys of Aldehyde of mAb thiol, aldehyde BMPH (8.1 Å)EVADE ribonuclease heterobifunctional EMCH (11.82 Å) KMUH (19 Å) M₂C₂HMPBH (17.9 Å) thiol of Cys of Amine of antibody Thiol, amine PDPH EVADEribonuclease heterobifunctional cleavable linker

A cleavable cross-linker of particular interest is made by incorporationof a protease sensitive peptide into the cross-linker. The protease, insome embodiments, is selected from a wide variety of naturally occurringenzymes, including endosomal and lysosomal proteases. Cathepsin B (alysosomal cysteine protease of the papain family) expression is elevatedin some cancerous cells, especially at the invasive edge of the tumor.Cathepsin B preferentially cleaves the Arg-Arg dipeptide, but ispromiscuous in its substrate recognition. Furin is a cellularendoprotease that catalyzes the proteolytic maturation of proteins inthe secretory pathway. Furin localizes predominately to the trans golginetwork, but does travel to many cellular compartments, includingendosomes, lysosomes, secretory granules, and the cell surface.

An alternative strategy is to use a cross linker that is sensitive toβ-lactamase and administer the β-lactamse concomitantly with thetargeted EVADE ribonuclease. There are several additional types oflinkers that can be cleaved such as peptide bonds, disulfide bonds,hydrazones, and phosphodiesters. The present invention is not limited tothe linkers discussed herein. One skilled in the art will appreciatethat a variety of linkers will find use with the present invention.

Conjugating Antibodies:

Many different antibodies may be conjugated the EVADE ribonuclease togenerate conjugates of the present invention. The CEA antigen is one ofmany known protein antigens that are over-expressed on the surface ofcancer cells and has been used previously to create targetedtherapeutics. Another antigen is CD33, which is present on acute myeloidleukemia cells. Acute myeloid leukemia (AML) is a cancer that may betreated by an anti-CD33 strategy. MYLOTARG is a CD33antibody-calicheamicin conjugate approved for treatment of AML. CD22 isa cell surface receptor found on B-cells which can also be used forantibody-based therapeutics.

The targeted EVADE ribonucleases may also be made using antibodiesagainst other cancer cell antigens. A variety of antibodies may beamenable to such a conjugation strategy. Among these, the preferredantigens of interest are:

-   -   over-expressed on cancer cells relative to normal cells    -   internalized by the cell (to facilitate ribonuclease entry into        the cytosol)    -   recognized by a monoclonal antibody

The following examples describe conjugates of humanized M195 (huM195),an antibody specific for CD33 (Immunotoxin Resistance in MultidrugResistant Cells. Cancer Res., 2003, 63, 72-79.) and an EVADEribonuclease QBI-50112 (R4C, L86E, N88C, R91D, V118C RNase I). Thelinkers are varied and include stable and cleavable linkers.

Non-Cleavable Linkers

Maleimide-Hydrazine

Carbohydrates found in the constant region of an antibody is oxidized toprovide an aldehyde, which is reactive with hydrazine. The hydrazine ofa cross-linker (BMPH, KMUH) is reacted with the aldehydes (oxidizedcarbohydrates) to form a hydrazone. The modified antibody then displaysa maleimide, which is reacted with the free thiol in a protein to forman antibody-protein conjugate. Thioether formation takes place atneutral pH.

The carbohydrates of huM195 are oxidized with by treatment of theantibody with 10 mM sodium periodate at room temperature forapproximately one hour at 4° C. The reaction is performed in the darkbecause sodium periodate is light sensitive. A desalting column(Amersham Biosciences, Sephadex G-25 Fine) is used prior to use ofoxidized huM195 for conjugation. A solution of BMPH is added to theoxidized huM195, and the reaction allowed to proceed for 30-60 minutesat room temperature. The reaction is then applied to a desalting column(Amersham Biosciences, Sephadex G-25 Fine)

Reaction times, ratios of reagents, solution concentrations, andtemperatures may be optimized to increase yield and purity of theconjugate. Fractions are collected, and their absorbance at 280 nmmonitored. Once the maleimide-huM195 containing fractions are pooled, asolution of the EVADE ribonuclease variant QBI-50112 (20 mM sodiumphosphate buffer, 0.15 M NaCl, pH 7.0 (PBS) with 10 mM EDTA) with a freecysteine residue is added in a one to one ratio with maleimide-huM195.The reaction is allowed to proceed for 30 minutes and then quenched bythe addition of Tris buffer with cysteine. The conjugated sample isapplied to a desalting column (Amersham Biosciences, Sephadex G-25 Fine)and eluted with buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.4).The fractions are monitored using the 280 nm absorbance. Theproduct-containing (huM195-QBI-50112) fractions are pooled. Reactiontimes, ratios of reagents, solution concentrations, and temperatures maybe optimized to increase yield and purity of the conjugate. A. B.

A. N-(β-maleimidopropionic acid) hydrazide · TFA (BMPH; 8.1 Å)B. N-(κ-maleimidoundecanoic acid) hydrazide (KMUH; 19.0 Å)Maleimide-NHS

The activated ester (an N-hydroxy succinimide) can be selectivelyreacted with amines in the antibody (typically lysine side chains)without affecting the maleimide. A covalent, chemically stable amidebond is formed. The modified antibody is then reacted with the singlefree thiol in the ribonuclease variant to form the conjugate. Thereaction of the thiol of the ribonuclease variant with the maleimide ismost selective at pH 6.5-7.5.

A four-fold excess of the crosslinker (EMCS or SMCC; Pierce) isdissolved in DMF (or DMSO if necessary) and is then added to a solutionof huM195 in buffer (20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0(PBS)). The reaction is allowed to proceed for 30 minutes at 4° C. Thereaction mixture is applied to a desalting column (Amersham Biosciences,Sephadex G-25 Fine). Fractions are collected, and their absorbance at280 nm monitored. Once the maleimide-huM195 containing fractions arepooled, a solution of the EVADE ribonuclease variant QBI-50112 (20 mMsodium phosphate buffer, 0.15 M NaCl, pH 7.0 with 10 mM EDTA) with afree cysteine residue is added in a one to one ratio withmaleimide-huM195. The reaction is allowed to proceed for 30 minutes andthen quenched by the addition of Tris buffer with cysteine. Theconjugated sample is applied to a desalting column (AmershamBiosciences, Sephadex G-25 Fine) and eluted with buffer (10 mM sodiumphosphate, 150 mM NaCl, pH 7.4). The fractions are monitored using the280 nm absorbance. The product-containing (huM195-QBI-50112) fractionsare pooled. Reaction times, ratios of reagents, solution concentrations,and temperatures may be optimized to increase yield and purity of theconjugate. A. B.

A. N-(ε-maleimidocaproyloxy) succinimide ester (EMCS; 9.4 Å)B. Succinimidyl 4-(N-maleimido-methyl)cyclohaxane-1-carboxylate (SMCC;11.6 Å)α-Haloacetyl-NHS

The activated ester (an N-hydroxy succinimide) is selectively reactedwith amines without affecting the haloacetyl. The optimal pH for thereaction is pH 7-9. The modified antibody is then reacted with thesingle free thiol in the ribonuclease variant to form the conjugate.

A solution of crosslinker (SBAP or SLAB) in DMSO is added to huM195solution (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2) and allowed toreact for approximately 30 minutes. The reaction mixture is be run overa desalting column (Amersham Biosciences, Sephadex G-25 Fine) withborate buffer (50 mM sodium borate, pH 8.3, 5 mM EDTA).

Fractions are collected, and their absorbance at 280 nm monitored. Asolution of the EVADE ribonuclease variant QBI-50112 (R4C, L86E, N88C,R91D, V118C RNase I) is added, and the reaction of the single free thiolof the RNase with the haloacetyl sits for approximately one hour. Thesereactions are performed in the dark due to the potential for sideproducts. The reactions are quenched by the addition of Tris buffer withcysteine. The quenching reaction is allowed to proceed for 15 minutes atroom temperature in the dark. The conjugated sample is applied to adesalting column (Amersham Biosciences, Sephadex G-25 Fine) and elutedwith buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.4 (PBS)). Thefractions are monitored using the 280 nm absorbance. Theproduct-containing (huM195-QBI-50112) fractions are pooled. Reactiontimes, ratios of reagents, solution concentrations, and temperatures maybe optimized to increase yield and purity of the conjugate. A. B.

A. Succinimidyl 3-(bromoacetamido) propionate (SBAP; 6.2 Å)B. N-Succinimidyl(4-iodoacetyl)aminobenzoate (SIAB; 10.6 Å)Cleavable Linkers

These linkers are peptide-based and are be cleaved by a human protease.The example describes linkers cleaved by the protease furin. Furinrecognizes Arg-Xaa-Yaa-Arg, where Xaa is unspecified and Yaa is Lys orArg. Hydrolysis occurs after the C-terminal Arg.

The reactive groups (maleimide, hydrazine, N-hydroxy succinimide ester,α-halo acetyl) used in the protease-sensitive cross-linkers are the sameas the commercially available linkers.

Peptides are dissolved in ddH₂O and added at a 3-fold molar excess to asolution of huM195 (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2) andallowed to react for approximately 30 minutes. The reaction mixture willbe run over a desalting column (Amersham Biosciences, Sephadex G-25Fine) with borate buffer (50 mM sodium borate, pH 8.3, 5 mM EDTA).Fractions are collected, and their absorbance at 280 nm monitored. Asolution of the EVADE ribonuclease variant QBI-112 in 20 mM sodiumphosphate, pH 7.0, containing NaCl (0.15 M) and EDTA (0.01 M) is added.The reaction proceeds at room temperature with stirring. Under theseconditions, reaction between the maleimide and a thiol is favored by1000-fold over reaction between the maleimide and an amine. After 30min, the reaction will be quenched by addition of a Tris-HCl buffercontaining cysteine at a concentration 10-fold greater than that of thepeptide substitutent. Peptides bearing the α-bromo-acetyl group areconjugated by using identical procedures, with three exceptions.Reactions are run in a 20 mM MOPS buffer, pH 8.2, containing NaCl (150mM) and EDTA (0.1 mM) for 60 min at 37° C. in the dark. The conjugatedsample is applied to a desalting column (Amersham Biosciences, SephadexG-25 Fine) and eluted with buffer (10 mM sodium phosphate, 150 mM NaCl,pH 7.4). The fractions are monitored using the 280 nm absorbance. Theproduct-containing (huMI95-QBI-50112) fractions are pooled. Reactiontimes, ratios of reagents, solution concentrations, and temperatures maybe optimized to increase yield and purity of the conjugate. A. D.

B

C.

A. Furin-sensitive maleimide-hydrazine cross-linkerB. Furin-sensitive maleimide-succinimide cross-linkerC. Furin-sensitive α-bromo acetyl-succinimide cross-linkerD. Identity of the R groups to be used in peptides A-C.Fusion Proteins

The cDNA encoding an anti-CEA scFv fragment will be fused to the 5′-endof the cDNA encoding the EVade ribonuclease QBI-50110 (R4C, L86E, N88R,G89D, R91D, V118C RNase I) RNase. Six glycine residues will beincorporated between the scFv and the ribonuclease to minimizehindrance. The cDNA will be sequenced, cloned in E. coli, and expressedas an insoluble protein sequestered in inclusion bodies. The inclusionbodies will be denatured and refolded. After refolding, the fusion willbe purified by standard protein chromatography methods, includingsize-exclusion and hydrophobic interactions. Production of the anti-CEAscFv-EVade fusion may be optimized to increase yield and/or purity.

The following reference, incorporated herein by reference in theirentireties, describe various ribonucleases, targeting moieties,conjugation methods, pharmaceutical compositions, and treatment methodsthat find use in conjunction with the present invention: U.S. Pat. Nos.3,627,876, 4,331,764, 4,882,421, 4,904,469, 5,200,182, 5,270,204,5,286,487, 5,286,637, 5,359,030, 5,389,537, 5,484,589, 5,529,775,5,540,925, 5,559,212, 5,562,907, 5,595,734, 5,660,827, 5,702,704,5,728,805, 5,786,457, 5,840,296, 5,840,840, 5,866,119, 5,955,073,5,973,116, 6,045,793, 6,051,230, 6,083,477, 6,175,003, 6,183,744,6,197,528, 6,235,313, 6,239,257, 6,271,369, 6,280,991, 6,290,951,6,312,694, 6,395,276, 6,399,068, 6,406,897, 6,416,758, 6,423,515,6,541,619, 6,558,648, 6,649,392, 6,077,499, 4,888,172, 6,653,104, andU.S. Pat. Publ. Nos. US20020006379A1, US20020037289A1, US20020048550A1,US20020111300A1, US20020119153A1, US20020187153A1, US20030031669A1,US20030219785A1, US20020106359A1, and US20030148409A1.

Example 2 Tumor Growth Inhibition by QBI-119

This Example describes the use of RNase variant QBI-119 for tumor growthinhibition. QBI-119 is an RNA variant that is R4C, G38R, R39D, L86E,N88R, G89D, R91D, V118C RNase I. The in vivo efficacy of this RNasevariant was determined using a standard xenograft model. In particular,A549 non-small human cancer cells (American Type Culture CollectionNumber CRL-1687) and Bx-PC-3 pancreatic cells (American Type CultureCollection Number CCL-185) were employed with athymic nude mice.Approximately 2×10⁶ cells were implanted into the right rear flank of5-6 week old male homozygous (nu/nu) nude mice (Harlan). Tumors wereallowed to grow to an average size of ≧75 mm³ before treatments wereinitiated. Animals of each tumor type, with the properly sized tumors,were divided into treatment groups, including one set of animals treatedwith vehicle (PBS) on the same dosing schedule as the treatment arm.QBI-119 was dosed at 15 mg/kg qdx5 (five times per week) throughout thecourse of the study. Tumors were measured twice weekly using calipers.Tumor volume (mm³) was determined by using the formula for an ellipsoidsphere: ${volume} = \frac{l \times w^{2}}{2}$

The percent tumor growth inhibition is determined using the followingformula: Percent  tumor  growth  inhibition  (%  TGI)${\%\quad{TGI}} = {1 - {\frac{\left( {{{final}\quad{size}} - {{starting}\quad{size}}} \right)_{treated}}{\left( {{{final}\quad{size}} - {{starting}\quad{size}}} \right)_{control}} \times 100}}$

The results of this Example are shown in FIGS. 1-2. FIG. 1A shows thetumor growth inhibition caused by QBI-119 on A549 cells was significant(64%), while FIG. 1B shows that QBI-119 had no significant impact onanimal weights at this dosage. FIG. 2A shows the tumor growth inhibitioncaused by QBI-119 on Bx-PC-3 cells was significant (60%), while FIG. 1Bshows that QBI-119 had no significant impact on animal weights at thisdosage.

All publications and patents mentioned in the above specification areherein incorporated by reference as if expressly set forth herein.Various modifications and variations of the described method and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in relevant fields areintended to be within the scope of the following claims.

Amendments to the Specification

On page 36, please replace the paragraph beginning on line 25 and endingon page 38, line line 1 with the following amended paragraph: Amino acidsequence for bovine ribonuclease A (SEQ ID NO: 1) 1 10 20 30 40KETAAAKFE RQHMDSSTSA ASSSNYCNQM MKSRNLTKDR CKPVNTFVHE 50 60 70 80 90SLADVQAVCS QKNVACKNGQ TNCYQSYSTM SITDCRETGS SKYPNCAYKT 100 110 120TQANKHIIVA CEGNPYVPVH FDASV

Amino acid sequence for human pancreatic ribonuclease (SEQ ID NO:2) 1 1020 30 40 KESRAKKFQ RQHMDSDSSP SSSSTYCNQM MRRRNMTQGR CKPVNTFVHE 50 60 7080 90 PLVDVQNVCF QEKVTCKNGQ GNCYKSNSSM HITDCRLTNG SRYPNCAYRT 100 110 120SPKERHIIVA CEGSPYVPVH FDASVEDST Amino Acid Three Letter AbbreviationsSingle Letter Abbreviations Alanine Ala A Arginine Arg R Asparagine AsnN Aspartic acid Asp D Cysteine Cys C Glutamine Gin Q Glutamic acid Glu EGlycine Gly G Histidine His H Isoleucine Lie I Leucine Leu L Lysine LysK Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser SThreonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V QBI-50101N88C RNase I QBI-50109 L86E, N88R, G89D, R91D RNase I QBI-50110 R4C,L86E, N88R, G89D, R91D, V118C RNase I QBI-50111 L86E, N88C, R91D RNase IQBI-50112 R4C, L86E, N88C, R91D, V118C RNase I QBI-50118 R4C, N88C,V118C RNase I QBI-50125 K7A, L86E, N88C, R91D RNase I QBI-50126 K7A,L86E, N88R, G89D, R91D RNase I QBI-50127 R4C, K7A, L86E, N88C, R91D,V118C RNase I QBI-50128 R4C, K7A, L86E, N88R, G89D, R91D, V118C RNase I

Please insert the attached Sequence Listing into the specification afterthe abstract.

1. A composition comprising a non-natural human ribonuclease one (humanRNase I) conjugated to a cell- or disease-specific targeting moiety,wherein said ribonuclease is configured to kill said cell or degradepathogenic RNA.
 2. The composition of claim 1, wherein said non-naturalhuman ribonuclease one has a variant sequence that disrupts binding tothe ribonuclease inhibitor.
 3. The composition of claim 1, wherein saidnon-natural human ribonuclease one has a variant sequence compared to anatural ribonuclease one selected from the group consisting of:N88C/L86E, N88R, G89D, R91D/R4C, L86E, N88R, G89D, R91D, V118C/L86E,N88C, R91D/R4C, L86E, N88C, R91D, VI 18C/R4C, N88C, V118C/K7A, L86E,N88C, R91D/K7A, L86E, N88R, G89D, R91D/R4C, K7A, L86E, N88C, R91D,V118C/R4C, G38R, R39D, L86E, N88R, G89D, R91D, V118C/and R4C, K7A, L86E,N88R, G89D, R9lD, V118C.
 4. The composition of claim 1, wherein saidcell is a cancer cell.
 5. The composition of claim 1, wherein saidpathogenic RNA is of viral origin.
 6. The composition of claim 1,wherein said ribonuclease is conjugated to said cell- ordisease-specific targeting moiety by a linker.
 7. The composition ofclaim 6, wherein said linker is attached to a non-native cysteine ofsaid ribonuclease.
 8. The composition of claim 1, wherein said targetingmoiety comprises an immunoglobulin.
 9. The composition of claim 8,wherein said immunoglobulin comprises a human or humanized antibody. 10.The composition of claim 8, wherein said immunoglobulin comprises anantibody fragment.
 11. The composition of claim 1, wherein saidtargeting moiety comprises a receptor ligand.
 12. The composition ofclaim 1, wherein said targeting moiety comprises a small molecule. 13.The composition of claim 12, wherein said small molecule comprises alipid or carbohydrate.
 14. The composition of claim 1, wherein saidtargeting moiety comprises an engineered non-natural protein.
 15. Thecomposition of claim 1, wherein said targeting moiety comprises apolymer.
 16. The composition of claim 1, wherein said targeting moietyis conjugated to said ribonuclease within a loop region of saidribonuclease corresponding to amino acids 84-95 of bovine ribonucleaseA.
 17. The composition of claim 1, wherein said ribonuclease and saidtargeting moiety comprise a fusion protein.
 18. A composition comprisinga nucleic acid molecule that encodes the composition of claim
 1. 19. Amethod for killing a cell comprising the step of exposing a cell to thecomposition of claim
 1. 20. The method of claim 19, wherein said cell isa cancer cell.